SUMMARY Pseudoexfoliation syndrome is a common, age-related systemic disorder of the extracellular matrix with important clinical ramifications including a particularly severe and common type of glaucoma, as well as cardiovascular diseases such as cardiomyopathy and aortic aneurysms. It is estimated that PEX syndrome may affect between 10 and 20 percent of people over the age of 60, and is responsible for over 25 percent of all open-angle glaucomas worldwide, accounting for the majority of glaucoma in some countries. Currently there are no experimental models, neither in vivo nor in vitro, capable of consistently replicating relevant characteristics of the disease such as the production of PEX material. Lack of such models represents an important problem because it limits our ability to test hypotheses on the cellular and molecular mechanisms of the disease, investigate how specific polymorphisms and gene-environment interactions might contribute to pathogenesis, and test experimentally promising new approaches such as genome editing and small molecules capable of inhibiting the production of PEX material. The overall objective of this application is to optimize a new experimental model that replicates relevant aspects of PEX syndrome in vitro and generate the tools needed to facilitate the use of this model by investigators in PEX research in the near future. Our central hypothesis is that the abnormal matrix metabolism characteristic of PEX syndrome can be replicated in vitro by combining the main pathophysiologic factors known to be associated with the disease; (I) genetic polymorphisms (using cells from PEX donors); (II) specific cell types known to produce abundant PEX material (using disease-relevant cells differentiated from induced pluripotent stem cells (iPSCs)); (III) aging (cellular senescence); and (IV) chronic exposure to additional environmental stressors and pro-fibrotic factors associated with PEX syndrome. To test this hypothesis we propose the following specific aims: 1) To differentiate iPSCs from PEX and control donors into smooth muscle cells (SMCs) and trabecular meshwork cells (TMCs), and optimize the culture conditions that best recapitulate the characteristics of PEX syndrome in vitro. 2) To generate conditionally immortalized SMCs and TMCs cell lines from PEX and control donors, and evaluate their potential as models of PEX syndrome. Completion of his project will open new opportunities in PEX research that are not currently possible by providing a complete set of new tools (immortal cell lines, experimental conditions to model PEX, and methods for quantification of PEX characteristics) that will allow reproducible testing of specific mechanistic hypothesis regarding the role of gene-environment interactions and epigenetic modifications in the pathogenesis of PEX, as well as systematic screening for potential therapeutic agents.